This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding plant sinapoylglucose:malate sinapoyltransferase (SMT) and its use in the conjugation of small molecules for materials.
Recent advances in genetic engineering have enabled the development of new biological platforms for the production of molecules, heretofore only synthesized by chemical routes. Although advances in fermentation technology have resulted in the use of microorganisms for the production of pharmaceutically useful proteins (antibiotics, enzymes etc.), the possibility of using green plants for the manufacture of high volume materials is becoming increasingly more attractive.
There are two obvious advantages of using green plants to produce large amounts of compounds that are traditionally synthetically manufactured. First, plants are a renewable energy resource. The photosynthetic ability of green plants means that the only raw materials that are required to produce carbon-based compounds in plants are CO2, water and soil nutrients. Second, in contrast to microbial fermentation, green plants represent a huge biomass that can easily accommodate the large amounts of chemicals that are required for high-volume, low-cost applications. The use of plants as production platforms for materials is complicated only in that they comprise a vastly more differentiated and complex genetic and biochemical systems as compared with microbes. Thus, production of molecules and materials from plants will be greatly enhanced if the materials to be produced are native, at least in some amounts to the plant.
Two classes of materials that are native to plants are aromatic acids and aromatic esters. In particular, p-hydroxybenzoic acid (pHBA) and esters of pHBA can readily be found. Both of these materials find use in various polymers useful in paints and other coatings. In addition, pHBA is the key monomer in Liquid Crystal Polymers (LCPs) which contain approximately 67% pHBA. Esters of pHBA can be used as backbone modifiers in other condensation polymers, i.e., polyesters, and are also used to make parabens preservatives.
It is known that aromatic acids, aromatic esters and pHBA are endogenous to plants as well as other organisms. In most bacteria, the generation of pHBA occurs by way of chorismate, an important branchpoint intermediate in the synthesis of numerous aromatic compounds, including phenylalanine, tyrosine, p-aminobenzoic acid and ubiquinone. In E. coli, chorismate itself undergoes five different enzymatic reactions to yield five different products, and the enzyme that is ultimately responsible for the synthesis of pHBA is chorismate pyruvate lyase, which is also known as CPL. The latter is the product of the E. coli ubiC gene, which was independently cloned by two different groups (Siebert et al., FEBS Lett 307:347-350 (1992); Nichlols et al., J. Bacteriol 174:5309-5316 (1992)). In higher plants the biosynthetic pathway leading to pHBA in Lithospermum erythrorhizon is thought to consist of up to ten successive reactions (L{haeck over (s)}scher and Heide, Plant Physiol. 106:271-279 (1992)), presumably all catalyzed by different enzymes.
Recently it has been shown that levels of pHBA production in plants may be enhanced through genetic manipulation. Several recent publications (Severin et al., Planta Medica, (1993) Vol. 59, No. 7, pp. A590-A591; Siebert et al., Plant Physiol. 112:811-819 (1996); WO 9600788), including Applicants own work (U.S. Ser. No. 09855,341) have demonstrated that tobacco plants (Nicotiana tabacum) transformed with a constitutively expressed chloroplast-targeted version of E. coli CPL (referred to as xe2x80x9cTP-UbiCxe2x80x9d) have elevated levels of pHBA that are at least three orders of magnitude greater than wildtype plants. However, it should be noted that these studies indicated that virtually all of the pHBA was converted to its two glucose conjugates, a phenolic glucoside and an ester glucoside. The conversion of the glucoside to a useful product will require a chemical step and represents an obstacle for the production of free pHBA or other aromatic acids. Therefore, a method of further processing the pHBA glucosides is needed.
There are no reports of endogenous plant transconjugation reactions that involve the transfer of benzoic acids from glucose esters to organic acids. However, there are reports of the processing of esters of hydroxycinnamic acids such as sinapic acid to malate conjugates as a function of secondary metabolism in cotyledon and leaf tissues of cruciferous plant species. Sinapic acid is generated from phenylalanine through the action of phenylalanine ammonia lyase (PAL) cinammate-4-hydroxylase, coumarate-3-hydroxylase, caffeic acid o-methyltransferase and ferulate-5-hydroxylase. Sinapoyl glucose is synthesized from sinapic acid and uridinediphosphate glucose (UDPG) through the action of UDPG sinapoyltransferase (SGT). Sinapoyl glucose is subsequently translocated to the vacuole. Sinapoyl glucose is a 1-O-glucose ester that has a free energy of hydrolysis (Mock and Strack, Phytochemistry 32:575-579 (1993)). This linkage provides the necessary free energy for the transacylation reaction catalyzed by sinapoylglucose:malate sinapoyltransferase (SMT) (Strack, Planta 155:31-36 (1982)), which generates sinapoyl malate in the expanding cotyledons (Sharma and Strack, Planta 163:563-568 (1985)). It is instructive to note that sinapoyl malate accumulated in the vacuole in these plants, although little is known about how vacuolar transport might be effected (Sharma and Strack (1985), supra). During seed maturation, sinapic acid is converted to sinapoyl choline by the combined actions of SGT and sinapoylglucose:choline sinapoyltransferase (SCT) (Strack et al., Z Naturforsch 38c:21-27 (1983)). Recently SMT has been partially characterized (Graewe et al., Planta 187(2):236-41 (1992)). However, despite the detailed biochemical understanding of these enzymes, none of the genes involved had been cloned, and relatively little is known about their regulation. Additionally, it is unclear how or if this enzymatic system may be adapted to the processing of benzoic acid glucosides and related molecules.
The problem to be solved therefore is to design a system for the production of benzoic acid derivatives and particularly pHBA derivatives in plants. Applicants have solved the stated problem by the discovery that sinapoylglucose:malate sinapoyltransferase (SMT) has the ability to convert glucosides of p-hydroxybenzoic acid to its corresponding malate conjugate where the malate product is localized in the plant vacuole. This further processing of the native p-hydroxybenzoic acid glucoside advances the art of materials production from genetically modified green plant platforms.
The present invention provides a method for the production of malate conjugated aromatic acids comprising: contacting a glycosylated aromatic acid with an effective amount of sinapoylglucose:malate sinapoyltransferase which catalyzes the substitution of a glucose moiety on the glycosylated aromatic acid with a malate moiety to form a malate conjugated aromatic acid. Suitable aromatic acids are described by the formula 
wherein
R1-R6 are each independently H, or OH, or COOH or OR7 or R7COOH and R7 is C1 to C20 substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;
providing at least one of R1-R6 is COOH
In an alternate embodiment the invention provides a method for the production of carboxylic acid conjugated aromatic acids comprising: contacting a glycosylated aromatic acid with an xcex1-hydroxycarboxylic acid of the general formula:
Rxe2x80x94COOH, where R is C1 to C20 substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;
and an effective amount of sinapoylglucose:malate sinapoyltransferase which catalyzes the substitution of a glucose moiety on the glycosylated aromatic acid with the xcex1-hydroxycarboxylic acid to form a carboxylic acid conjugated conjugated aromatic acid.
In another embodiment the invention provides a method for the production of aromatic esters comprising:
contacting a glycosylated aromatic acid with an alcohol of the general formula:
Rxe2x80x94OH, where R is C1 to C20 substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;
and an effective amount of sinapoylglucose:malate sinapoyltransferase to form an aromatic ester.
Preferred aromatic acids of the invention include para-hydroxybenzoic acid. Preferred xcex1-hydroxycarboxylic acids of the invention include lactate. Preferred alcohols of the invention include methanol, ethanol and isopropanol.
In a preferred embodiment the invention provides a method for the production of pHBA malate comprising a) providing a host cell producing suitable levels of glycosylated pHBA; b) introducing into the host cell a nucleic acid molecule encoding sinapoylglucose:malate sinapoyltransferase; wherein the sinapoylglucose:malate sinapoyltransferase catalyzes the substitution of a glucose moiety on the glycosylated pHBA with a malate moiety to form pHBA malate.